Hemorrhagic fever viruses have been discussed in the scientific literature. The following publications, patents, and patent applications are cited in this application as superscript numbers:    1. Charrel, R. N. and de Lamballerie X., ANTIVIRAL RESEARCH. 57:89-100 (2003).    2. Peters C. J., “Arenavirus diseases,” in Porterfield J., ed., EXOTIC VIRAL INFECTION, London: Chapman and Hall Medical, 227-246 (1995).    3. Buchmeier, M. J., Clegg, J. C. S., Franze-Fernandez, M. T., Kolakofsky, D., Peters, C. J., and Southern, P. J., “Virus Taxonomy: Sixth Report of the International Committee on Taxonomy of Viruses,” Murphy, F. A., Fauquet, C. M. et al., Eds. Sprnger-Verlag, New York, 319-323 (1995).    4. Clegg, J. C. S., Bowen, M. D., et al., “Arenavirideal” in Van Regenmortel, M. H. V., Fauquet, C. M., Bishop, D. H. L., Carsten, E. B., Estes, M. K., Lemon, S. M., Maniloff, J., Mayo, M. A., McGeoch, D. J., Pringle, C. R., Wickner, R. B. (Eds) Virus Taxonomy. Seven Report of the International Committee for the Taxonomy of Viruses, Academic Press, New York, pp 633-640 (2000).    5. McCormick, J. B., Epidemiology and control of Lassa fever, CURR. TOP. MICROBIOL. IMMUNOL., 134: 69-78 (1987).    6. Leifer, E., Gocke, D. J., et al., Report of a laboratory-acquired infection treated with plasma from a person recently recovered from the disease, AM. J. TROP. MED. HYG., 19:677-679 (1970).    7. McCormick, J. B., King, I. J., Webb, P. A., et al., Lassa Fever: Effective therapy with Ribavirin, N. ENGL. J. MED., 314: 20-26 (1986).    8. Kilgore, P. E., Ksiazek, T. G., Rollin, P. E., et al., Treatment of Bolivian Hemorrhagic Fever with intravenous ribavirin, CLIN. INFECT. PIS., 24: 718-722 (1997).    9. Enria, D. A., and Maiztegui, J. I., Antiviral treatment of Argentine Hemorrhagic Fever, ANTIVIRAL RES., 23: 23-31 (1994).    10. Huggins, J. W., Prospects For Treatment Of Viral Hemorrhagic Fevers With Ribavirin, A Broad-Spectrum Antiviral Drug, REV. INFECT. DIS., II: Suppl. 4:S750-S761 (1989).    11. Candurra, N. A., Maskin, L., and Pamonte, E. B., Inhibition of arenavirus multiplication in vitro byphenotiazines, ANTIVIRAL RES., 31(3): 149-158 (1996).    12. Glushakova, S. E., Lakuba, A. I., Vasiuchkov, A. P., Mar'iankova, R. F., Kukareko, T. M., Stel'makh, T. A., Kurash, T. P., and Lukashevich, I. S., Lysosomotropic agents inhibit the penetration of arenavirus into a culture of BHK-21 andvero cells, VOPROSY VIRUSOLOG II. 35(2): 146-150 (1990).    13. Petkevich, A. S., Sabynin, V. M., Lemeshko, N. N., Lukashevich, I. S., and Beloruss, N., Study of the effect of rimantadine on the reproduction of several arenaviruses, EPIDEMIOL. MIKROBIOL., 138-143 (1982).    14. Wachsman, M. B., Lopez, E. M. F., Ramirez, J. A., Galagovsky, L. R., and Coto, C. E., Antiviral effect of brassinosteroids against herpes virus and arenavirus, ANTIVIRAL. CHEM. CHEMOTHER., 11(1): 71-77 (2000).    15. Rawls, W. E., Banerjee, S. N., McMillan, C. A., and Buchmeier, M. J., Inhibition of Pichinde virus replication by actinomycin D, J. GEN. VIROL., 33(3): 421-434 (1976).    16. Enria, D. A., Feuillade, M. R., Levis, S., Briggiler, A. M., Ambrosio, A. M., Saavedra, M. C, Becker, J. L., Aviles, G., Garcia, J., Sabattini, M., “Impact of vaccination of a high-risk population for Argentine hemorrhagic fever with a live-attenuated Junin virus vaccine” in Saluzzo, J. F., Dodet, B., (eds) FACTORS IN THE EMERGENCE AND CONTROL FOR RODENT-BORNE VIRAL DISEASES, Paris: Elsevier, 1999, pp. 273-279 (1999).    17. Bagai, S. and Lamb, R. A., J. CELL BIOL., 135: 73-84 (1996).    18. Beyer, W. R., et al., J. VIROL., 77: 2866-72 (2003).    19. Bowen, M. D., et al., VIROLOGY, 219: 285-90 (1996).    20. Castagna, A., et al., DRUGS, 65: 879-904 (2005).    21. Childs, J. E., and Peters, C. J., “The Arenaviridae” Ed Salvato (ed.), Plenum Press, New York, pp. 331-84 (1993).    22. Cianci, C., et al., ANTIMICROB AGENTS CHEMOTHER, 48: 2448-54 (2004).    23. Clegg, J. C., CURR TOP MICROBIOL IMMUNOL, 262: 1-24 (2002).    24. Froeschke, M., et al., J. BIOL CHEM., 278: 41914-20 (2003).    25. Garcia, C. C., et al., ANTIVIR CHEM CHEMOTHER, 11: 231-7 (2000).    26. Hall, W. C., et al., AM J TROP MED HYG, 55: 81-8 (1996).    27. Harman, A., et al., J VIROL, 76: 10708-16 (2002).    28. Jeetendra, E., et al., J VIROL, 77: 12807-18 (2003).    29. Kinomoto, M., et al., J Virol, 79: 5996-6004 (2005).    30. Kunz, S., et al., VIROLOGY, 314: 168-78 (2003).    31. Lenz, O., et al., PROC NATL ACAD SSI USA, 98: 12701-5 (2001).    32. Maron, M. D. and Ames, B. N., MUTAT RES, 113: 173-215 (1983).    33. Oldfield, V., et al., DRUGS, 65: 1139-60 (2005).    34. Peters, C. J., et al., CURR TOP MICROBIOL IMMUNOL, 134: 5-68 (1987).    35. Petkevich, A. S., et al., VOPR VIRUSOL, 244-5 (1981).    36. Southern, P. J., VIROLOGY, 2: 1505-51 (2001).    37. Weissenbacher, M. C., et al., INFECT IMMUN, 35: 425-30 (1982).    38. West, J. T., et al., J VIROL, 75: 9601-12 (2001).    39. Yao, Q. and Compans, R. W., J VIROL, 69: 7045-53 (1995).    40. Beyer, W. R., et al., “Oncoretrovirus and lentivirus vectors pseudotyped with lymphocytic choriomeningitis virus glycoprotein: generation, concentration, and broad host range.” J. VIROL. 76:1488-1495 (2002).    41. Connor, R. I., et al., “Vpr is required for efficient replication of human immunodeficiency virus type-1 in mononuclear phagocytes.” VIROLOGY 206:935-944 (1995).    42. Naldini, L., et al., “In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.” SCIENCE 272:263-267 (1996).    43. Rojek, J. M., et al., “Characterization of the cellular receptors for the South American hemorrhagic fever viruses Junin, Guanarito, and Machupo.” VIROLOGY 349:476-491 (2006).    44. Simmons, G., et al., “Characterization of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) spike glycoprotein-mediated viral entry.” PROC. NATL. ACAD. SCI. USA 101:4240-4245 (2004).    45. Wool-Lewis, R. J., and P. Bates, “Characterization of Ebola virus entry by using pseudotyped viruses: identification of receptor-deficient cell lines.” J. VIROL. 72:3155-3160 (1998).
All of the publications, patents, and patent applications cited in this application are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety.
The National Institute of Allergy and Infectious Diseases (NIAID) and the Centers for Disease Control and Prevention (CDC) have classified a number of viruses as potential agents of bioterrorism (www.bt.cdc.gov/agent/agentlist-category.asp). The highest threat agents, the Category A pathogens, have the greatest potential for adverse public health impact and mass casualties if used in ill-intentioned ways. Within the Category A pathogens, there are a number of viruses that can cause viral hemorrhagic fevers with high case fatality rates. The Category A hemorrhagic fever viruses pose serious threats as potential biological weapons because: 1) they can be disseminated through aerosols; 2) a low dose (1-10 plaque forming unit (pfu)) can cause disease; 3) they cause severe morbidity and mortality (case fatality rates of 15-30%); 4) they can cause fear and panic in the general public; 5) there are no U.S.-approved effective vaccines or specific antivirals available; 6) these pathogens are easily available and can be readily produced in large quantities; and 7) research on weaponizing various hemorrhagic fever viruses has been conducted.1 
Arenaviruses are enveloped viruses with a genome that consists of two single-stranded RNA segments designated small (S, 3.5 Kb) and large (L, 7.5 Kb), both with an ambisense coding arrangement.36 The S RNA segment encodes the major structural proteins, nucleocapsid protein (NP) and a precursor envelope protein (GPC) encoding two envelope glycoproteins (external GP1 and transmembrane GP2),18, 24, 30, 31 and the L RNA segment encodes the RNA polymerase protein L and an 11 KDa protein, Z protein, with putative regulatory function.19 GP1 and GP2, which form the tetrameric surface glycoprotein spike, are responsible for virus entry into targeted host cells.
The family Arenaviridae consists of a single genus (Arenavirus) that includes several viruses (currently 23 recognized viruses1) causing severe hemorrhagic fever diseases in humans.2 The Arenaviridae family has been divided into two groups is according to sequence-based phylogeny. The “Old World” group, originated from Africa, includes the human pathogens lymphocytic choriomeningitis (LCM) virus and Lassa virus. The “New World” group, originated from Latin America, is divided into 3 clades. Clade B includes, in addition to Tacaribe and Amapari viruses, the Category A human pathogenic viruses Junin (Argentine hemorrhagic fever), Machupo (Bolivian hemorrhagic fever), Guanarito (Venezuelan hemorrhagic fever), and Sabiá (Brazilian hemorrhagic fever). These Category A viruses are capable of causing severe and often fatal hemorrhagic fever disease in humans.
Rodents are the natural host of arenaviruses, although Tacaribe virus is found in bats. The arenaviruses characteristically produce chronic viremic infections in their natural host,15 which in turn shed virus in their urine and feces, ultimately infecting humans in close contact with these infected materials either by aerosol or direct contact with skin abrasions or cuts. The natural history of the human disease is determined by the pathogenicity of the virus, its geographical distribution, the habitat and the habits of the rodent reservoir host, and the nature of the human-rodent interaction.21 
Several Arenaviruses are associated with severe hemorrhagic disease in human. Lassa virus (from the Old World group) is responsible for Lassa hemorrhagic fever, while 4 viruses from the New World group (all from Clade B) cause severe hemorrhagic fever in human. Those viruses are: Junin virus responsible for Argentine hemorrhagic fever, Machupo virus for Bolivian hemorrhagic fever, and Guanarito virus for Venezuelan hemorrhagic fever. Sabia virus was isolated from a fatal case of hemorrhagic fever in Brazil. It is estimated that Lassa virus causes 100,000-300,000 infections and approximately 5,000 deaths annually.5 So far, an estimated 30,000 confirmed cases of Junin infections have been documented, while about 2,000 of Machupo, 200 of Guanarito and only 2 of Sabia.1 
Recent concerns over the use of Arenaviruses as biological weapons have underscored the necessity of developing small molecule therapeutics that target these viruses.1 The availability of antiviral drugs directed at these viruses would provide treatment and a strong deterrent against their use as biowarfare agents. Since antiviral drugs can be easily administered (oral, pill, or liquid) and exert their antiviral effect within hours of administration, they will serve to effectively treat diseased patients, protect those suspected of being exposed to the pathogen (post-exposure prophylaxis), and assist in the timely containment of an outbreak.
Currently, there are no virus-specific treatments approved for use against Arenavirus hemorrhagic fevers. Present disease management consists of general supportive care: monitoring and correcting fluid, electrolyte and osmotic imbalances and treating hemorrhaging with clotting factor or platelet replacement. Convalescent immune serum therapy may be effective in treating cases of Junin and Machupo virus disease, but the availability of such serum is extremely limited.
Ribavirin, a nucleoside analog, has been used with some success in Lassa fever patients. In small trials, intravenous ribavirin given to patients within the first 6 days after development of fever decreased mortality from 76% to 9%.7-9 A controlled trial of 18 patients with Argentine hemorrhagic fever resulted in 13% mortality in treated patients compared with 40% in untreated patients.10 Ribavirin therapy is associated with adverse effects including a dose-related, reversible hemolytic anemia and also has demonstrated teratogenicity and embryo lethality in several animal species. It is therefore classified as a pregnancy category X drug, contraindicated during pregnancy. Intravenous ribavirin is available in limited supplies in the U.S. for compassionate use under an FND application. The dosing regimen for ribavirin therapy that has been used in cases of Lassa fever consists of an initial 30 mg/kg intravenous (IV) loading dose, followed by 16 mg/kg IV every 6 hours for 4 days; then 8 mg/kg IV every 8 hours for 6 days (total treatment time 10 days). The cost of treatment for an adult male is approximately $800. The attributes of ribavirin make it less than ideal for the treatment of Arenavirus hemorrhagic fevers.
A number of in vitro inhibitors of Arenavirus replication have been reported in the literature including phenothiazines, trifluoroperazine and chlorpromazine,1 amantadine,12,13 brassinosteroids14 and actinomycin D.15 The anti-Arenavirus activities of these compounds are generally weak and non-specific.
The only Arenavirus hemorrhagic fever for which studies have been undertaken toward development of a vaccine has been Argentine hemorrhagic fever (AHF) caused by Junin virus. A live-attenuated vaccine, called Candid 1, has been evaluated in controlled trials among agricultural workers in AHF-endemic areas, where it appeared to reduce the number of reported AHF cases with no serious side effects.16 It is not known if the Candid 1 vaccine would be useful against other Arenavirus hemorrhagic fevers and this vaccine is not available in the United States of America.
Tacaribe virus is a biosafety level 2 (BSL 2) New World arenavirus (NWA) that is found in Clade B and phylogenetically related to the Category A NWA (Junin, Machupo, Guanarito and Sabiá). Tacaribe virus is 67% to 78% identical to Junin virus at the amino acid level for all four viral proteins. In order to screen for inhibitors of NWA a high-throughput screening (HTS) assay for virus replication was developed using Tacaribe virus as a surrogate for Category A NWA. A 400,000 small molecule library was screened using this HTS assay. A lead series was chosen based on drug properties and this series was optimized through iterative chemistry resulting in the identity of a highly active and specific small molecule inhibitor of Tacaribe virus with selective activity against human pathogenic NWA (Junin, Machupo, Guanarito and Sabiá). This molecule demonstrates favorable pharmacodynamic properties which permitted the demonstration of in vivo anti-arenavirus activity in a newborn mouse model.
All human pathogens Arenaviruses from the New World group causing hemorrhagic fever are from the Clade B. These human pathogen viruses require manipulation under high-level containment (BSL-4). However, Amapari and Tacaribe viruses also from Clade B can be grown in tissue culture under BSL-2 (low-level) containment. Working under low-level containment makes experimentations easier and safer with these viruses. While Amapari virus produces low cytopathic effect, Tacaribe virus can be grown readily in cell culture and produce robust CPE in 4 to 6 days. Since this CPE is directly related to viral replication, compounds that inhibit virus replication in cell culture can be identified readily as conferring protection from virus-induced CPE (although it is theoretically possible to inhibit CPE without inhibiting virus replication). Moreover, compounds having identified activity against Tacaribe virus will also likely be active against Arenavirus human pathogen causing hemorrhagic fever (Junin, Machupo, Guanarito and Sabia) given the high degree of homology (around 70% identity for all 4 proteins of Tacaribe virus compared to Junin virus, with long stretch of protein with perfect identity) between these viruses.
What is needed in the art are new therapies and preventives for the treatment of viral infections and associated diseases, such as caused by hemorrhagic fever viruses like Arenaviruses.